MONOMERIC ISOMERS OF HUMAN INTERLEUKIN-5 SHOW THAT 1 1-RECEPTOR RECRUITMENT IS SUFFICIENT FOR FUNCTION/

The normally dimeric human interleukin 5 (IL-5) was re-engineered into two monomeric Isomer forms to investigate mechanistic features of rec eptor recognition, One form, denoted GM1-IL-5, is a CD-loop expanded f orm, in which an 8-residue linker designed for flexibility was inserte d between residues 85 and 86, The second, denoted DABC-IL-5, is a circ ularly permuted form of human IL-5 in which a chain discontinuity was introduced in the CD loop and the two consequent chain fragments were joined at the normal N and C termini by a di-glycyl linker, Both IL-5 isomers folded into stable monomers in solution as shown by sedimentat ion equilibrium and CD and formed an intrachain disulfide bond predict ed from the structure of wild type IL-5, From titration microcalorimet ry and optical biosensor analyses, both monomers were shown to interac t with tile IL-5 receptor alpha chain with 1:1 stoichiometry and affin ities 30- to 40-fold weaker than for the dimeric wild type protein, An d both monomers stimulated cell proliferation of human IL-5 receptor p ositive cells with a concentration dependence close to that of wild ty pe, The data show that both monometric and dimeric forms of IL-5 funct ion through similar 1:1 receptor alpha chain recruitment processes and that if: is the helical packing of the monomeric four-helix bundle un it in IL-5, rather than the helical connectivity itself, that appears to play the major role in presenting structural epitopes to trigger fu nctional receptor activation.